The process by which human cytomegalovirus (HCMV) capsids exit the nucleus (nuclear egress) is incompletely understood. Viral DNA synthesis, capsid assembly, and packaging (encapsidation) are thought to occur in sub-nuclear structures called replication compartments (RCs), after which capsids must reach the inner nuclear membrane for their release into the cytoplasm. How capsids move to the nuclear periphery is unresolved, although an actomyosin-based transport mechanism has been suggested for other herpesviruses. The HCMV nuclear egress complex (NEC) is comprised of inner nuclear membrane-bound UL50, and its soluble binding partner, UL53, both of which are essential for nuclear egress. While certain NEC functions require complex formation at the inner nuclear membrane, there is evidence that UL53 and its homologs participate in upstream processes. Our preliminary data support a model whereby UL53 links capsids to myosin Va in RCs to facilitate nuclear F-actin dependent capsid transport to the nuclear periphery. While we have made progress in our studies, we have yet to definitively show whether UL53, nuclear F-actin, and myosin Va are involved in active capsid trafficking per se, or whether UL53 directly links capsids to myosin Va to mediate this process. We are also interested in whether capsid transport initiation is coordinated with upstream processes in RCs, for which we have supporting data that we wish to explore further. In Aim 1 of this proposal we will (1) assess whether HCMV capsids undergo active transport to the nuclear periphery using live cell imaging and particle tracking analysis, and then use this system to interrogate whether deletion of UL53, depolymerization of nuclear F-actin with drug-treatment, and inhibition of myosin Va with dominant negative mutants similarly hinder capsid trafficking, and (2) use biochemical approaches to assess whether UL53 physically links capsids to myosin Va to direct this process. In Aim 2, we will (1) explore whether capsid transport is spatially coordinated with encapsidation at specific sites in RCs using TEM of cryosubstituted serial sections and immunoEM, and (2) further apply these techniques to examine whether UL53 and myosin Va engage DNA-filled C capsids to initiate their transport from these locations. Together, these results may illustrate that UL53 acts in a continuum during nuclear egress by directing capsid movement from RCs to the nuclear periphery, a mechanism that may be conserved among herpesviruses. Since HCMV is a clinically relevant pathogen that causes birth defects in newborns and life-threatening complications in immunocompromised individuals, there is a considerable need for additional therapeutic interventions. A better understanding of nuclear egress could not only inform HCMV drug development, but may also uncover novel features of host cell biology.